INHIBITION OF ENZYMES 823 



to normal values it is likely that (1) some inactivation has occurred, (2) in- 

 sufficient time has been provided for the reversal, (3) the binding of the 

 mercurial to the enzyme is much tighter than to the reversor (K^"^ K^), 



or (4) some secondary factor has complicated the situation, e.g., failure to 

 add a displaced cofactor (page 1-625) or the oxidation of enzyme SH groups 

 by the oxidized thiol reversor (page 1-625). If the complete reversibility 

 of the inhibition has been established, and if (E^) and K^ are known, it is 

 possible to calculate the value of K^ from experiments in which low con- 

 centrations of reversor are added and the ij determined. 



It is relatively easy to treat equilibrium conditions quantitatively, but 

 the problems encountered in considering the rates of reversal are at the 

 present mainly insoluble. Investigators have been chiefly interested in whe- 

 ther reversal occurs or not and since extremely few rate studies have been 

 made, there are not adequate data upon which to base accurate analyses. 

 If the reversal occurs in two steps — the dissociation of EI and the combi- 

 nation of I with R — the individual reactions are not as simple as with most 

 inhibitions. For example, at physiological pH and with most media used, 

 the dissociation of EI must be written as: 



EI + H+ + X- -► EH + IX (7-20) 



where X~ represents any complexing anion in the medium. Likewise, the 

 second step must usually be described by: 



IX + RH -> RI + X- + H+ (7-21) 



Now it is not known if the reactions occur in this manner, or whether I 

 can be directly transferred from E to R without forming the IX complex, 

 either on the surface of the enzyme or in solution. A study of the effects 

 of (X^) on the reversal rate would help to solve this problem. It is also 

 not known if the reactive form of the reversor is RH or R~, but from the 

 effects of pH on the rates of reaction of mercurials with simple thiols it 

 appears that at least the form R" reacts much more rapidly than RH, as 

 would be anticipated. In any event, the pH must be an important factor 



